The general objective of the proposed research is to investigate the binding of estrogens, androgens, and corticoids to the proteins in plasma which bind them. We will use affinity chromatography to isolate enough human corticosteroid-binding globulin (CBG) to ascertain its amino acid sequence. In an effort to gain some information about the nature of the cortisol-binding site in CBG we will conduct experiments in which we will attempt to place an affinity label in or close to the binding site of the purified protein. In vitro perfusion of human placenta will be conducted with unbound cortisol, cortisol bound to CBG, and cortisol bound to albumin, to examine the effect of these parameters on cortisol transport across the placenta both from mother to fetus and vice versa. Purified rat CBG, also isolated by affinity chromatography, will be used to investigate the effect of CBG on cortisol action in the rat in vivo. We will continue our efforts to isolate human testosterone- estradiol-binding globulin. This will involve a continuation of our studies on agents which denature this protein and agents which protect it from denaturation. We plan to use affinity chromatography as the main purification procedure and in conjunction with this we are in the process of developing a novel way in which to link steroid derivatives to Sepharose 4B. This procedure involves the coupling to Sepharose 4B of a diamino compound, azodianiline, which contains an internaazo bond susceptible to dithionite cleavage. It will allow the steroid-TeBG complex to be removed intact from the Sepharose.